Human-murine chimeric antibodies against respiratory syncytial virus

ABSTRACT

This invention relates to a human antibody which contains the one CDR from each variable heavy and variable light chain of at least one murine monoclonal antibody, against respiratory syncytial virus which is MAb1129 and the use thereof for the prevention and/or treatment of RSV infection.

This application is a continuation-in-part of U.S. application Ser. No.07/813,372, filed on Dec. 23, 1991, now abandoned.

BACKGROUND

Respiratory syncytial virus (RSV) is the major cause of acuterespiratory illness in young children admitted to hospitals, and thecommunity practice will treat perhaps five times the number ofhospitalized children. It is therefore, the most common cause of lowerrespiratory tract infection in young children. While the majority ofcommunity-acquired RSV infections resolve themselves in a week to tendays, many hospitalized children, especially under six months of agerequire assisted ventilation.

Efforts to produce an effective vaccine have been unsuccessful (8). Amajor obstacle to vaccine development is safety; the initial formalininactivated RSV vaccine caused an increased incidence of RSV lowerrespiratory tract disease and death in immunized children upon exposureto virus (5).

Recently, the drug ribavirin has been licensed for therapy of RSVpneumonia and bronchiolitis (2,3); its value is controversial (4).Although ribavirin has shown efficacy (9), the drug has to beadministered over an 18 hour period by aerosol inhalation. In addition,the level of secondary infections following cessation of treatment issignificantly higher than in untreated patients.

Studies have shown that high-titered RSV immunoglobulin was effectiveboth in prophylaxis and therapy for RSV infections in animal models (6,7). Infected animals treated with RSV immune globulin, showed noevidence of pulmonary immune-complex disease (6, 7).

Even if RSV hyperimmune globulin is shown to reduce the incidence andseverity of RSV lower respiratory tract infection in high risk children,several disadvantages may limit its use. One drawback is the necessityfor intravenous infusion in these children who have limited venousaccess because of prior intensive therapy. A second disadvantage is thelarge volume of RSVIG required for protection, particularly since mostthese children have compromised cardiopulmonary function. A thirddisadvantage is that intravenous infusion necessitates monthly hospitalvisits during the RSV season which places these children at risk ofnosocomial RSV infection (1). A final problem is that it may prove to bevery difficult to select sufficient donors to produce a hyperimmuneglobulin for RSV to meet the demand for this product. Currently onlyabout 8% of normal donors have RSV neutralizing antibody titers highenough to qualify for the production of hyperimmune globulin.

Another approach may be the development of monoclonal antibodies withhigh specific neutralizing activity as an alternative to hyperimmuneglobulin. It is preferable, if not necessary, to use human monoclonalantibodies rather than murine or rat antibodies to minimize thedevelopment of human anti-rodent antibody responses which may compromisethe therapeutic efficacy of the antibody or induce immune-complexpathology. However, the generation of human monoclonal antibodies withthe desired specificity may be difficult and the level of productionfrom human cell lines is often low, precluding their development.

An alternative approach involves the production of human-mouse chimericantibodies in which the genetic information encoding the murine heavyand light chain variable regions are fixed to genes encoding the humanheavy and light constant regions. The resulting mouse-human hybrid hasabout 30% of the intact immunoglobulin derived from murine sequences.Therefore, although a number of laboratories have constructed chimericantibodies with mouse variable and human constant domains (10-18), themouse variable region may still be seen as foreign (19).

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide acomplementarity determining region (CDR)-grafted human antibody whichcontains at least one CDR from each variable heavy chain and variablelight chain of at least one monoclonal antibody, against the RSVantigen. The monoclonal antibody may be derived from any non-humananimal, preferably however, it is derived from a rodent and mostpreferably it is a murine monoclonal antibody. Preferably, the murinemonoclonal antibody is a neutralizing antibody. It is also preferablethat said murine antibody is an antibody against RSV F antigen.

The term "animal" as used herein is used in its broadest sense includesmammals including humans.

DETAILED DESCRIPTION OF THE DRAWINGS

The drawings depicted and described herein are intended to furtherillustrate the present invention and are not intended to limit theinvention in any manner whatsoever.

FIG. 1 shows the amino acid (AA) sequence design of CDR-Grafted anti-RSVF glycoprotein V_(H). The figure depicts the AA sequence for the humanHV3 V_(H) before grafting (SEQ ID NO:16), CDR grafted V_(H) (SEQ IDNO:17), and murine MAb1308F V_(H) (SEQ ID NO:18) from which the CDRsequence was grafted. The heavily underlined regions identify the CDRsequence which was grafted into the human HV3 V_(H) and each of thethree regions is identified as CDR1, CDR2 and CDR3, respectively.

FIG. 2 shows the amino acid (AA) sequence design of CDR-Grafted anti-RSVF Protein V_(L). The figure depicts the AA sequence for the human K102V_(L) before grafting (SEQ ID NO:19), CDR grafted V_(L) (SEQ ID NO:20),and murine MAb1308F V_(L) (SEQ ID NO:21) from which the CDR sequence wasgrafted. The heavily underlined regions identify the CDR sequence whichwas grafted into the human K102 V_(L) and each of the three regions isidentified as CDR1, CDR2 and CDR3, respectively.

FIG. 3 depicts the oligonucleotides used to make Hu1308V_(H), thesequences which are underlined are the specific primer sequences (SEQ IDNO:22-25).

FIG. 4 depicts the oligonucleotides used to make Hu1308V_(L), thesequences which are underlined are the specific primer sequences (SEQ IDNO:26-29).

FIG. 5 depicts the plasmid construction of the expression vectors forHumanized 1308.

FIG. 6 depicts a graph of the Neutraliziation of RSV as percentneutralization versus ng MAb per reaction for neutralizing with CosHu1308F and with Mu1308F.

FIG. 7 shows the amino acid (AA) sequence design of CDR-Grafted anti-RSVF glycoprotein V_(H). The figure depicts the AA sequence for the humanCOR V_(H) before grafting (SEQ ID NO:30), CDR grafted V_(H) (SEQ IDNO:31), and murine MAb1129 V_(H) (SEQ ID NO:32) from which the CDRsequence was grafted. The heavily underlined regions identify the CDRsequence which was grafted into the human COR V_(H) and each of thethree regions is identified as CDR1, CDR2 and CDR3, respectively.

FIG. 8 shows the amino acid (AA) sequence design of CDR-Grafted anti-RSVF Protein V_(L). The figure depicts the AA sequence for the human K102V_(L) before grafting (SEQ ID NO:33), CDR grafted V_(L) (SEQ ID NO:34),and murine MAb1129 V_(L) (SEQ ID NO:35) from which the CDR sequence wasgrafted. The heavily underlined regions identify the CDR sequence whichwas grafted into the human K102 V_(L) and each of the three regions isidentified as CDR1, CDR2 and CDR3,respectively.

FIG. 9 shows the oligonucleotides used to construct the humanized 1129 V(SEQ ID NO:36-42).

FIG. 10 shows binding data for humanized 1129 in an ELISA assay.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have found that transplantation into a human antibody, ofonly the genetic information for at least one CDR from each of thevariable heavy and variable light chain derived from murine monoclonalantibody against RSV antigen, is effective for the prevention andtreatment of RSV in animals. Preferably the murine antibody is aneutralizing antibody against RSV. Another aspect of the presentinvention provides for the murine antibody to be an antibody against RSVF antigen. Preferably, the murine antibody is neutralizing antibodyagainst RSV F antigen. The substitution of the mouse CDR's into thehuman variable framework segments minimizes the potential for humananti-mouse antibody (HAMA) responses while retaining binding affinityand specificity for antigen, RSV F protein. Since, the CDR's do notcontain characteristic murine or human motifs, the human antibodiescontaining the murine antibody CDR's are essentially indistinguishablefrom completely human antibodies, thereby, minimizing the human antibodyresponse while retaining binding affinity and specificity for RSV Fantigen.

The development of a humanized antibody against RSV F antigen began witha murine antibody against RSV F antigen. Examples of murine antibodiesof this type are: MAb 1436C, MAb 113, MAb 112, MAb 151, MAb 1200, MAb1214, MAb 1237, MAb 1129, MAb 1121, MAb 1107, MAb 131-1, MAb 43-1, MAb1112, MAb 1269, MAb 1243, MAb 1331H, MAb 1308F and MAb 1302A (seecitation 21).

An aspect of the present invention provides that the CDRs of the humanantibody are comprised of three complementarity determining regions(CDRs) from each variable heavy and variable light chain of the murineantibody.

The murine antibodies against RSV F antigen have been mapped bycompetitive binding and reactivity profiles of virus escape mutants tothree broad antigenic sites (A, B, C) containing 16 distinct epitopes(20). The epitopes within antigenic sites A and C have shown the leastvariability in natural isolates.

Therefore, another aspect of this invention provides for a humanantibody containing at least one CDR from each variable heavy andvariable light chain of at least one murine antibody against RSV Fantigen which is specific for antigenic site A or C. In one aspect, thisinvention provides for the murine antibody against RSV F antigenspecific for antigenic site C, where the murine antibody is MAb 1308F.

In such an embodiment of this invention a human antibody contains CDR'sof the variable heavy chain of murine antibody MAb 1308F against the RSVF antigen. The CDR variable heavy chain of MAb 1308F comprises threeCDRs having the following amino acid sequences: Nos. 31 to 35, 47 to 60and 99 to 106. In addition, this embodiment contains CDR's of a variablelight chain of MAb 1308F of murine antibody against RSV F antigen. TheCDR variable light chain comprises three CDR's having the followingamino acid sequences: Nos. 24 to 34, 50 to 56 and 89 to 97.

Another aspect of this invention provides for a human antibodycontaining at least one CDR from each variable heavy and variable lightchain of at least one murine antibody against RSV F antigen which isspecific for antigenic site C. Preferably, this invention provides forthe murine antibody against RSV F antigen specific for antigenic site C,where the murine antibody is MAb 1129.

In the embodiment of this invention a human antibody which containsCDR's of the variable heavy chain of murine antibody MAb 1129 againstthe RSV F antigen. The CDR variable heavy chain of MAb 1129 comprisesthree CDRs having the following amino acid sequences: Nos. 31 to 36, 52to 67 and 100 to 109. In addition, this embodiment contains CDR's of avariable light chain of MAb 1129 of murine antibody against RSV Fantigen. The CDR variable light chain comprises three CDR's having thefollowing amino acid sequences: Nos. 24 to 33, 51 to 56 and 89 to 96.

An additional aspect of applicants' invention is a process forpreventing or treating RSV infection comprising administering to theanimal an effective amount of a human antibody containing at least oneCDR from each variable heavy and variable light chain, of at least onemurine antibody against RSV F antigen.

Another aspect of applicants' invention is a composition comprisingadministering an effective amount of the human antibody as describedabove in conjunction with an acceptable pharmaceutical carrier.Acceptable pharmaceutical carriers include but are not limited tonon-toxic buffers, fillers, isotonic solutions, etc.

The composition of Applicant's invention may be administered topicallyor systemically. Examples of topical administration are intranasaladministration and inhalation of an aerosol containing the humanantibody composition. Systemic administration may be accomplished byintravenous or intramuscular injection of the human antibodycomposition.

A preferred aspect of Applicants' invention is that the human antibodyis administered as part of a plurality of human antibodies against RSV Fantigen. These antibodies can be against the same or different epitopesof the RSV F antigen.

Additionally, the human antibody of this invention can be usedclinically for diagnosing respiratory syncytial virus in patients.Because of their affinity for RSV F antigen these human antibodies canbe used in known diagnostic assay procedures for detecting the presenceand concentration of RSV F antigen cells in samples, e.g., body fluids.The human antibodies of the present invention can for example beattached or bound to a solid support, such as latex beads, a column,etc., which are then contacted with a sample believed to contain RSV Fantigen.

Applicants' development of human antibodies against RSV, began withmurine hybridoma cells producing murine monoclonal antibodies which havebeen shown to neutralize RSV in vitro and protect cotton rats againstlower respiratory tract infection with RSV.

One such antibody was selected, which is specific for antigenic site C,to produce mouse-human chimeric antibodies. This antibody was chosen onthe basis that it: (i) reacted with a large number of virus strainstested (at least 13 out of 14 isolated); (ii) retained neutralizingactivity against virus escape mutants selected with other anti-Fantibodies and (iii) blocked RSV replication when administered at lowdoses to cotton rats by intranasal route prior to virus challenge. Theantibody showed significant reduction in pulmonary virus titer amongantibodies in that respective region. Murine antibody 1308F, specificfor the C region of RSV F protein, was chosen as the initial target forhumanization.

In summary, the human antibodies were constructed as follows: the RNAwas extracted from the murine antibody-producing cell line, the murinevariable regions which are responsible for the binding of the antibodyto RSV were cloned and sequenced, resulting in the identification of themurine antibody CDRs. Then a human variable heavy and light chainframework sequence having the highest homology with the variable heavyand light chain murine antibody, was selected. A human frameworksequence such as described above is best able to accept themurine-derived CDRs.

The murine 1308F variable heavy chain was compared to various humangermline genes, the highest homology was to the human germline gene HV3.The two sequences were 62% homologous overall and 65% in the frameworkregions. Significantly, there is good homology at the junctions of theCDR segments and the frameworks with the exception of the 5' end of FR2.The murine derived variable heavy chain CDRs were then substituted intothe variable heavy chain human germline gene HV3. The mouse and humansequences as well as that of a potential CDR-Grafted combination of thetwo is shown in FIG. 1.

A similar analysis of the V_(L) region revealed high homology to thehuman germ line V-Kappa gene K 102. The alignment of these sequences isshown in FIG. 2. In this case the homology is 62% overall and 73% in theframework regions. The murine-derived variable light CDRs were thensubstituted into the human variable light chain of human germline geneK102. In each case a human J-region can be selected which is identicalto the mouse sequence.

In another embodiment, murine 1129 variable heavy chain was compared tovarious human variable region amino acid sequences, the highest homologywas to the human rearranged COR sequence. The two amino acid sequenceswere 75% homologous overall and 80% in the framework regions.Significantly, there is good homology at the junctions of the CDRsegments and the frameworks. The murine derived variable heavy chainCDRs were then substituted into the variable heavy chain human COR V_(H)sequence. The mouse and human sequences as well as that of a potentialCDR-Grafted combination of the two is shown in FIG. 1.

A similar analysis of the V_(L) region revealed high homology to thehuman germ line K102. The alignment of these sequences is shown in FIG.8. In this case the homology is 73% overall and 82% in the frameworkregions. The murine-derived variable light CDRs were then substitutedinto the human variable light chain of human germline K102. In this casea human J-region, human JK4, was selected which is similar to the mousesequence.

Therefore, human antibodies are expressed and characterized relative tothe parental murine antibodies to be certain that the geneticmanipulation has not drastically altered the binding properties of theantibodies.

Applicants present herein examples which are further illustrative of theclaimed invention but not intended to limit the invention.

EXAMPLE 1

cDNA cloning and sequencing of anti-RSV F Protein antibody 1308F

cDNA copies of the V_(H) and V_(L) of the target antibody were generatedas follows. The first strand cDNA reaction was carried out using AMVreverse trenscriptase and a phosphorylated oligonucleotide primercomplementary to a segment of the mRNA coding for the constant region ofthe particular heavy or light chain isotype. For 1308F the isotype isgammal, kappa and the specific oligonucleotides were5'AGCGGATCCAGGGGCCAGTGGATAGAC (SEQ ID NO:1) complementary to codons129-137 of the CH1 region of the murine Gammal gene, and5'TGGATGGTGGGAAGATG (SEQ ID NO:2) complementary to codons 116-122 of themurine C-kappa gene. The primer anneals to a segment of the mRNAadjacent to the variable region. Second strand cDNA synthesis wascarried out using RNase H and E. coli DNA polymerase I, as described byGubler and Hoffman (Gene 25,;263, 1983), followed by T4 DNA polymeraseto assure that blunt ends are produced.

    ______________________________________                                        Signal      V      J       C     mRNA                                                     1st    strand  cDNA                                                           2nd    strand  cDNA                                               ______________________________________                                    

The ds-cDNA was ligated into pUC18 which had been digested withrestriction endonuclease SmaI and treated with alkaline phosphatase. Theligation was used to transform E. coli DH5a by the method of Hanahan (J.Mol. Biol. 166;557, 1983). Oligonucleotide probes corresponding toC-region sequence lying between the first strand cDNA primer and theV-region were used in colony hybridizations to identify transformantscarrying the desired cDNA segment. The specific probe sequences wereGGCCAGTGGATAGAC (SEQ ID NO:3) complementary to codons 121-125 of murineCH1 regions and TACAGTTGGTGCAGCA (SEQ ID NO:4) complementary to codons110-115 of c-Kappa, respectively. Candidate plasmids, isolated fromcolonies which were positive in the hybridization, were analyzed bydigestion with restriction endonucleases Eco RI and Hind III to releasethe CDNA insert. Those with inserts of 400-500 bp were subjected to DNAsequencing.

The cDNA inserts were inserted into M13 mp18 and mp19 for thedetermination of the DNA sequence on both strands. Single stranded DNAfrom the resulting recombinant bacteriophage was isolated and sequencedby the dideoxy chain termination method (Proc. Nat. Acad. Sci. USA 74;5463, 1977).

In order to confirm that the pair of rearranged and somatically mutatedV gene cDNA's isolated from the 1308F hybridoma represented those whichwere in the 1308F antibody, a single-chain Fv gene was generated,expressed in and secreted from mammalian cells, then assayed for bindingto RS virus. Competition binding experiments then were used todemonstrate the identity of the binding site.

EXAMPLE 2

Design and assembly of human 1308F V_(H) and V_(L)

The CDR regions of the V_(H) and V_(L) were identified by comparing theamino acid sequence to known sequences as described by Kabat (38). Inorder to select the human framework sequences best able to accept themouse derived CDR sequences in a conformation which retains thestructure of the antigen combining site, the following strategy wasemployed. First, the sequence of the murine V_(H) and V_(L) regions willbe compared to known human sequences from both the Genbank and NBRFprotein databanks using the Wordsearch program in the Wisconsin packageof sequence manipulation programs (Nucleic Acid Res. 12; 387). The bestseveral human V-regions were then analyzed further on the basis ofsimilarity in the framework regions, especially at the junctions of theframework and CDR regions (see FIGS. 1 and 2).

The CDR-grafted V_(H) region together with the respective leadersequence of the human v-region gene was synthesized de novo using fouroverlapping oligonucleotides ranging from 100-137 nucleotides in length(see FIG. 3). The oligonucleotides were first allowed to anneal inpairwise combinations and extended with DNA polymerase to generateapproximately 200 bp ds DNA fragments with an overlapping region. thefragments were then mixed and subjected to PCR using primers at the 3'end of one fragment and the 5' end of the other fragment. The onlyproduct which can be formed under these condition is the full lengthV_(H) segment. The specific primer sequences are underlined in FIG. 3.An endonuclease Sac I site was included at the 3' end of the V_(H)sequence in order to join it to a human constant region gene segment.

The CDR-grafted V_(L) region was synthesized in a similar way (see FIG.4). In this instance the initial 200 bp fragments were amplifiedseparately and inserted into separate plasmeds. The fragment coding forthe amino terminus was cloned into a pUC18 derivative as an NcoI-SmaIfragment while the fragment coding for the carboxyl-terminus was clonedas a SmaI to Hind III fragment. The fragments were subsequently combinedvia a SmaI site at the junction. The oligonucleotides are indicated inFIG. 4. A Hind III site was included near the 3' end of the gene segmentin order to join it to a human C-kappa gene.

EXAMPLE 3

Construction of Vectors for 1308F expression

The NcoI-SacI fragment representing the humanized V_(H) was joined to aSacI-Notl fragment representing a human c-Gamma I CDNA and inserted intopS 18 (which is pUC 18 with Ncol and NotI restriction sites incorporatedinto the polylinker region between the BamHI and Kpnl sites). Thehumanized 1308F-gammal gene on a SacI-NotI fragment was then combinedwith a Pvul-NotI fragment from pSJ37 carrying a poly A addition site anda PvuI-SacI fragment from pSV2-dhfr-pCMV containing the SV40 origin ofreplication, a dhfr gene and the CMV immediate early promoter. Theresulting plasmid was designated pSJ60.

The NcoI-HindIII fragment representing the humanized V_(L) was joined toa HindIII-Notl fragment representing a human c-Kappa CDNA in pS18. Thehumanized 1308F-Kappa gene on a SalI-NotI fragment was then combinedwith a Pvul-NotI fragment from pSJ37 carrying a poly A addition site anda PvuI-SalI fragment from pSV2-dhfr-pCMV, containing the SV40 origin ofreplication, a dhfr gene and the CMV immediate early promoter. Theresulting plasmid was designated pSJ61.

Finally pSJ60 and pSJ61 were combined into a single plasmid containingboth the light and heavy chains and expression signals. This wasaccomplished by isolating a PvuI-Bam HI fragment from pSJ61 carrying thelight chain with a PvuI-Bgl II fragment from pSJ60 carrying the heavychain to generate pSJ66. (See FIG. 5).

EXAMPLE 4

Transfection of Cosl cells with PSJ60 and PSJ61

Transfections were carried out according to the method of McCutchan andPagano (J. Nat. Can. Inst. 41: 351-356, 1968) with the followingmodifications. COS 1 cells (ATCC CRL1650) were maintained in ahumidified 5% C02 incubator in 75 cm² tissue culture flasks inDulbecco's Modified Eagle Medium (DMEM, GIBCO #320-1965) supplementedwith 10% Fetal Bovine Serum (FBS, GIBCO #200-6140) and 2 mM L-glutamine(BRL #320-5030) and passed at a split ratio of 1:20 when the cells hadreached confluence. 48 hours prior to transfection, 5 100 mm tissueculture dishes were seeded with 1.5×10⁶ cells per dish in 12 ml DMEM,10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin (P-S, GIBCO#600-5070). The day of the transfection, 120 ug each of the plasmidspSJ60 and pSJ61 were combined, ethanol precipitated, and asepticallyresuspended in 2.5 ml Tris-Buffered-Saline. The resuspended DNA wasadded dropwise, with mixing, to 10 ml of DMLEM containing 1 mg/mlDEAE-dextran (Phamiacia #17-0350-01) and 250 uM chloroquine (Sigma#C6628). The medium was removed from the COS1 cells in the 100 mm dishesand the cells were washed once with Dulbecco's phosphate buffered saline(D-PBS, GIBCO #310-4190), and 2.5 ml DMEM supplemented with 10% NuSerum(Collaborative Research #55000) were added to each plate. 2.5 ml of theDNA/DEAE-dextran/chloroquine mix were added dropwise to each plate, theplates swirled to mix the DNA, and were returned to the incubator. After4 hours in the incubator, the supernatant was aspirated from the cellsand the cells were washed once with 5 ml D-PBS. The cells were shockedfor 3 minutes by the addition of 5 ml of 10% dimethylsulfoxide (DMSO) inD-PBS at room temperature. The DMSO was aspirated from the cells and thecells were washed with 5 ml D-PBS. 14 ml of DMEM/10% FBS/2 mML-glutamine/1% P-S were added to each plate and the plates were returnedto the incubator.

Three days post-transfection the medium was removed from the plates,pooled, and stored at -20° C. The cells were harvested, pooled, andseeded into 4 150 cm² tissue culture flasks two with 40 ml DMEM/10%NuSerum and two with 40 ml DMEM/10% FBS/2 mM L-glutamine. The medium wascollected and the cells refed at 7, 10, and 14 days. In this way a totalof 125 ug of humanized 1308F antibody was accumulated in 310 ml ofmedium supplemented with FBS and 85 ug in 240 ml of medium supplementedwith NuSerum.

EXAMPLE 5

Transfections of COS 1 cells with PSJ66

48 hours prior to transfection, 5 100 mm tissue culture dishes wereseeded with 1.5×10⁶ cells per dish in 12 ml DMEM, 10% FBS, 2 mML-glutamine, 1% penicillin-streptomycin (P-S, GIBCO #600-5070). The dayof the transfection, 125 ug of the plasmid pSJ66 were ethanolprecipitated and aseptically resuspended in 1.0 ml Tris-Buffered-Saline.The resuspended DNA was added dropwise, with mixing, to 4.0 ml of DMEMcontaining 1 mg/ml DEAE-dextran (Pharmacia #17-0350-01) and 250 uMchloroquine (Sigma #C6628). The medium was removed from the COS1 cellsin the 100mm dishes and the cells were washed once with Dulbecco'sphosphate buffered saline (D-PBS, GIBCO #310-4190), and 2.5 ml DMEMsupplemented with 10% NuSerum (Collaborative Research #55000) were addedto each plate. 2.5 ml of the DNA/DEAE-dextran/chloroquine mix were addeddropwise to each plate, the plates swirled to mix the DNA, and werereturned to the incubator. After 4 hours in the incubator, thesupernatant was aspirated from the cells and the cells were washed oncewith 5 ml D-PBS. The cells were shocked for 3 minutes by the addition of5 ml of 10% dimethylsulfoxide (DMSO) in D-PBS at room temperature. TheDMSO was aspirated from the cells and the cells were washed with 5 mlD-PBS. 14 ml of DMEM/10% FBS/2 mM L-glutamine/1% P-S were added to eachplate and the plates were returned to the incubator.

Three days post-transfection the medium was removed from the plates,pooled, and stored at -20° C. The cells were harvested, pooled, andseeded into 4 150 cm² tissue culture flasks two with 40 ml DMEM10%NuSerum and two with 40 ml DMEM10% FBS/2 mM L-glutamine. The medium wascollected and the cells refed at 7, 10, and 14 days. In this way a totalof 190ug of humanized 1308F antibody was accumulated in 310 ml of mediumsupplemented with FBS and 120 ug in 240 ml of medium supplemented withNuSerum.

The concentration of humanized 1308F antibody secreted from the Coslcells into the medium was determined using a capture ELISA. Goatanti-human IgG Fc coated onto 96 well plates was used to capture thehumanized antibody. Peroxidase conjugated goat anti-human whole IgGdeveloped with a chromogenic substrate was then used to detect the boundantibody. A purified human IgG1/Kappa preparation was used to calibratethe assay.

EXAMPLE 6

Neutralization of RSV with humanized 1308F

METHODS:

RSV was neutralized with either humanized 1308F from Cos cellsupernatant or purified 1308F murine monoclonal antibody. This was doneby incubating 50 plaque-forming units of RSV with serial 2-folddilutions of antibody for 1.0 hour at 37° C. Confluent monolayers ofHep2 cells in 24 well panels were infected with 100 μl of antibodytreated virus, untreated control virus, and mock infected controls.Incubated for 1.5 hours at 37° C., humidified , tand 5% CO₂ andoverlayed with 1.5 mL EMEM, 1% FBS, and 1% methyl cellulose. Cells werefixed and stained with glutaldehyde and crystal violet on day 4. Plaqueswere counted in triplicate wells and plotted as percent neutralization.The results shown in FIG. 6 indicate that both the purified murine 1308Fmonoclonal and the humanized 1308F monoclonal antibody at 5 to 10 ng perwell yield similar 50% reductions in RSV plaques.

EXAMPLE 7

Generation of a CDR-grafted A-site antibody 1129

Poly-A+ RNA was purified from a lysate of 2×107 murine 1129 hybridomacells using oligo-dt cellulose. First strand CDNA was made from 1 ug pA+RNA using random hexamer primers and AMV reverse transcriptase" 1 ug pA+RNA, 50 mM Tris-HCl pH 8.5, 8 mM Mg₂ Cl, 30 mM KCl, 1 mM dithiothrietol,1 mM dNTP's, 25 units of placental ribonuclease inhibitor, 33 uM randomhexamer and 10 units of AMV reverse transcriptase for one hour at 42° C.The cDNA from the 1129 VL region was amplified by PCR usingoligonucleotides SJ41 and SJ11, see Table 1. cDNA from the 1129 VHregion was similarly amplified using oligonucleotides SJ42 and SJ10, seeTable 1.

                                      TABLE 1                                     __________________________________________________________________________    SJ10                                                                          AGCGGATCCAGGGGCCAGTGGATAGAC (SEQ ID NO: 1)                                    SJ11                                                                          GATGGATCCAGTTGGTGCAGCATC (SEQ ID NO: 5)                                       SJ41                                                                          CACGTCGACATTCAGCTGACCCAGTCTCCA (SEQ ID NO: 6)                                 SJ42                                                                          CGGAATTCAGGTIIAICTGCAGIAGTC(A,T)GG (SEQ ID NO: 7)                             (I = deoxy-Inosine)                                                           SJ53                                                                          CCCAAGCTTGGTCCCCCCTCCGAACGTG (SEQ ID NO: 8)                                   SJ154                                                                         GGCGTCGACTCACCATGGACATGAGGGTCC(C/T)CGCTCAGC (SEQ ID NO: 9)                    SJ155 (H1129L CDR 1)                                                          GTCACCATCACTTGCAAGTGCCAGCTGAGTGTAGGTTACATGCACTGGTACC                          AGCAG (SEQ ID NO: 10)                                                         SJ157 (H1129L CDR 3)                                                          GCAACTTATTACTGCTTTCAGGGGAGTGGGTACCCATTCACGTTCGGAGGGG                          GG (SEQ ID NO: 11)                                                            SJ168                                                                         GTGACCAACATGGACCCTGCTGATACTGCCAC (SEQ ID NO: 12)                              SJ169                                                                         CCATGTTGGTCACTTTAAGGACCACCTGG (SEQ ID NO: 13)                                 SJ170                                                                         CCAGTTTACTAGTGTCATAGATCAGGAGCTTAGGGGC (SEQ ID NO: 14)                         SJ171                                                                         TGACACTAGTAAACTGGCTTCTGGGGTCCCATCAAGG (SEQ ID NO: 15)                         __________________________________________________________________________

PCR conditions

0.5 uL of 1st strand CDNA, 10 mM Tris-HCl pH8.3, 50 mM KCl, 1.5 mMMg2Cl, 0.2 mM dNTP's, 0.001% gelatin, 1 uM each primer, 1 ng DNAtemplate and 2.5 u AmpliTaq(TM) DNA polymerase (Perkin Elmer--Cetus).94° 1 minute, 55° 2 minutes, 72° 2 minutes in Perkin Elmer 480thermocycler for 25 cycles. The resulting DNA fragment(s) were thenextracted once with phenol/chloroform (1/1), precipitated with 2.5volumes of ETOH, resuspended in the appropriate restriction endonucleasebuffer and digested with restriction endonucleases to produce cohesiveends for cloning. The resulting fragments were then separated byelectrophoresis on a 1% agarose gel. After staining the gel withethidium bromide the fragments were excised and purified from theagarose by freezing and extraction in the presence of phenol.

The fragments were then digested with restriction endonucleases EcoRland BamHl and cloned into plasmid pUC18. The inserts were then sequencedby the dideoxynucleotide chain termination method using modified T7 DNApolymerase (Seqeunase, US Biochemical). The translated sequences werecompared to human antibody protein sequences. The VL was found to bemost homologous to the K102 light chain and the VH was found to be mosthomologous to the Cor VH region. The 1129 Fv region was then modeled bysubstitution of the residues from the 1129 VL and VH sequence into thecoordinates of corresponding residues in the crystal structure theMCPC603 antibody. Residues were identified as being integral to thefolded structure or solvent exposed by visual inspection of the model.

Several residues which were integral and which were different in themouse and human sequences were left as the mouse residue in order tomaintain the integrity of the Fv and thus the binding site. Suchresidues were 31,83,113, and 116 on the VH and 47 in the VL region. Theresulting sequences are shown in FIGS. 7 and 8.

The designed humanized 1129 VH was constructed using syntheticoligonucleotides SJ147-SJ153 (FIG. 9) (SEQ ID NO:36-42) which werecombined using PCR. The products of this PCR were then digested withNcol and Sacl and cloned into pladmid vector pSJ40 which is a pUC18derivative in which an out of frame lacZ1 segment is restored in frameas a fusion to an in-frame V region segment when such a segment isinserted as an Ncol-Sacl fragment. A plasmid containing an insert inwhich 5 mutations were clustered in a single 50 bp region was thensubjected to repair of these changes using recombinant PCR and theprimers SJ168 and SJ169, see Table 1.

The VL was generated by site directed mutagenesis of the humanized 1308Flight chain gene. Oligonucleotides SJ155, see Table 1, (CDR1), and SJ157(CDR3) were used to separately mutagenize the H1308L gene. Mutagenesiswas carried out using T7 DNA polymerase on uracil containing singlestranded DNA templates generated in E. coli strain BW313 (dut-,ung-) andsubsequently transformed into E. coli strain DH5 (dut+,ung+). The twomutants were combined and CDR2 introduced by recombinant PCR usingoligonucleotides SJ170, SJ154, see Table 1, (5' end) and SJ171, SJ53,see Table 1, (3' end). The CDR-grafted VH and VL genes were placed intopSJ60 (see Example 3) and pSJ61 (see Example 3), respectively asNcol-Sacl fragments in place of the H1308F Vregion segments resulting inplasmids pSJ81 and pSJ105. In addition the murine VH and VL cDNAsegments were similarly joined to human C-Gammal and CKappa respectivelyto generate expression vectors pSJ75 and pSJ84.

EXAMPLE 8

Hu1129 Transient Expression

COS1 cells (ATCC CRL1650) were maintained in a humidified 5% CO₂incubator in 75 CM² tissue culture flasks in Dulbecco's Modified EagleMedium (DMEM, GIBCO #320-1965) supplemented with 10% fetal bovine serum(FBS, GIBCO #200-6140) and 2 mM L-glutamine (GIBCO #320-5030) and passedat a split ratio of 1:20 just prior to reaching confluence.

Transfections were carried out according to the method of McCutchan andPagano (J. Nat. Can. Inst. 41: 351-356, 1968) with the followingmodifications. Twenty four hours prior to transfection 100 mm tissueculture dishes (Corning #25020) were seeded with 2×106 COS1 cells perdish in 14 ml DMEM, 10% FBS, 2 mM L-glutamine. The day of thetransfection 10 ug of the Hu1129 heavy chain plasmid (pSJ81, fromExample 7 were combined with 10 ug of the Hu1129 kappa light chainplasmid pSJ105, from Example 7, the DNA was ethanol precipitated andaseptically resuspended in 1.0 ml Tris-Buffered-Saline. The resuspendedDNA was added dropwise, with mixing, to 4.0 ml of DMEM containing 1mg/ml DEAE-dextran (Pharmacia #170350-01) and 250 uM Chloroquine (Sigma#C6628). The medium was removed from the COS1 cell dishes, the cellmonolayers were washed once with 10 ml Dulbecco's phosphate bufferedsaline (D-PBS, GIBCO #310-4190), and 2.5 ml DMEM supplemented with 10%NuSerum (Collaborative Research #55000) and 2 mM L-glutamine were addedto each plate. 2.5 ml of the DNA/DEAEdextran/chloroquine mix were addeddropwise to each plate, the plates were swirled to mix the DNA, andreturned to the incubator. After an eight hour DNA adsorption period theplates were removed from the incubator and the supernatant was aspiratedfrom the plates. The cells were shocked by the addition of 5 ml of 10%DMSO in D-PBS per plate for 3 minutes at room temperature, after whichthe DMSO was aspirated from the cells and the cells were washed oncewith 5 ml D-PBS. 15 ml DMEM, 10% NuSerum, 2 mM L-glutamine (productionmedium) were added to each plate and the plates were returned to theincubator.

Seventy two hours post-transfection the conditioned medium was harvestedfrom the plates and stored at -20° C., andl 5 ml production medium wasadded to the plates and the plates were returned to the incubator.Ninety six hours later the medium was collected from the plates andstored at 20° C.

EXAMPLE 9

Quantitation of Hu1129

Quantitation of the Hu1129 IgGl antibody secreted into the medium by theCOS1 cells was performed using a sandwich type ELISA. In brief, NuncMaxisorp Immunoplates (Nunc #439454) were coated with 50 ul/well of 0.5ug/ml goat anti-human IgG Fc (Cappel #55071) in 0.1M sodium bicarbonatepH 9.6 for 3 hours at room temperature. The wells were washed threetimes with 0.01M sodium phosphate pH 7.4, 0.15M NaCl, 0.1% Tween 20(PBS-T). Nonspecific protein binding to the plate was blocked bytreatment of the wells with 200 ul/well of 3% (w/v) nonfat dry milk inPBS for 30 minutes at room temperature. A purified human IgGl kappastandard (Sigma #1-3889) was made up at 100 ng/ml in PBS-T and seriallydiluted 1:2 to 1.56 ng/ml, and 50 ul of each were added to duplicatewells of the assay plate. COS1 cell supernatants were diluted in PBS-Tand duplicate 50 ul samples were added to the plate. After an one hourroom temperature incubation the wells were evacuated and washed threetimes with PBS-T. To detect the presence of bound Hu1129 antibody,horseradish peroxidase conjugated affinity purified goat anti-human IgG(whole molecule, Cappel #3601-0081) was diluted 1:1000 in PBS-T and 50ul was added to each well of the assay plate and incubated at roomtemperature for one hour. The plate was washed three times with PBS-Tand 100 ul of the chromogenic substrate TMBlue (TSI #TM102) was added toeach well. The plate was incubated at room temperature in the dark forten minutes and the reaction was stopped by the addition of 50 ul perwell of 4.5M H₂ SO₄. The plate was read at 450 nm using a MolecularDevices Vmax microplate reader, and data analysis was performed usingSoftmax software (Molecular Devices) running on an IBM P/S2 model 80computer.

During the first seventy two hours of production the COS1 cells produced0.06 ug/ml Hu1129, for a total of 0.9 ug. In the next ninety six hoursof production the COS1 cells produced 0.99 ug/ml Hu1129, for a total of14.85 ug.

EXAMPLE 10

Hu1129 Binding Assay

Binding assays of the Hu1129 were performed in a capture ELISA,essentially as for the quantitation ELISA, but with the followingchanges. Plates were coated with the Mul 331 antibody at 0.5 ug/well,the wells were blocked with 3% non-fat milk in PBS-T, and 50 ul of RSVinfected HEP2 cell lysate was added to each well and incubated at roomtemperature for 1 hour. The remainder of the assay was carried out asfor the quantitation assay starting with the addition of diluted samplesto the wells. Results were analyzed as a double reciprocal plot of OD vsantibody concentration from which an apparent Kd for the H1129 moleculeof 0.7 nM was determined compared to 10 nM for the M1129HuGammal,Kappaantibody.

RSV neutralization assays on H1129 and ch1129 antibody were performedaccording to the following procedure:

1. Unwrap 96 well Costar cell culture plates in hood.

2. Warm Growth Medium (GM) to 37° C.

3. Thaw MA104 cells at 37° C. Dilute to ˜150,000 cells per mL with GM.Mix cells and dispense 200 μl per well.

4. Culture cells 37° C., 5% CO₂, and humidified overnight beforeinfection.

5. Dilute RSV Stock to 10,000 pfu per mL in Maintenance Medium (MM).

6. Mix equal volume of Antibody diluted in MM with equal volume ofdiluted RSV. Incubate at 37° C., 5% CO₂, and humidified for 1.0 h beforeinfection.

7. Infect replicate wells of MA104 cells with 200 μl of the Antibody andVirus mixture. Infect replicate wells with virus and mock infectedcontrols.

8. Wrap the plates in cellophane and incubate at 37° C., 95% humidity,and 5% CO₂ for 5 days.

9. ELISA for RSV: Aspirate each well; add 100 μl 80% Acetone/PBS(vol./vol.) and incubate at room temperature 30 minutes.

10. Aspirate each well and air dry for 30 minutes on the grill of alaminar flow hood.

11. Wash 4 times with PBS, 0.05% Tween 20.

12. Add 100 μl of monoclonal antibody to RSV F-protein to each well.Incubate for 1.0 h at 37° C.

13. Wash 4 times with PBS, 0.05% Tween 20.

14. Add 100 μl of anti-murine antibody goat serum-horse radishperoxidaze conjugate to each well. Incubate for 1.0 h at 37° C.

15. Wash 4 times with PBS, 0.05% Tween 20.

16. Add 100 μl of a freshly prepared 1:1 mixture of ABTS and peroxide toeach well. Incubate at room temperature until the optical density (405nm) of the virus control is 5 to 10 times that of the mock infectedcontrols.

Appendix:

Growth Medium (GM): Minimum Essential Medium (Eagle) with

Earle's BSS,

2 mM glutamine,

Eagle's non-essential amino acids 0.1 mM final,

Fetal bovine serum 10% (v/v),

Penicillin 50 units/ml,

Streptomycin 50 mcg/ml

Maintenance Medium (MM): as above with serum reduced to 1 to 2%.

MA104 cell stocks are grown up in T150 flasks with Growth Medium. Stocksare frozen at 3×10⁶ cells per 1.8 mL vial in 10% DMSO and Growth Medium.Stored in a LN₂ refrigerator.

RSV stocks: are grown up in MA104 (monkey kidney) or Hep 2 cells in T150flasks. Add ˜0.2 ml (˜100,000 pfu) virus stock per confluent T150.Adsorption for 1.0 h at room temperature. Then add 20 mL maintenancemedium with 1% fetal bovine serum. Incubate 4-5 days at 37° C. Collectcells just before 100% cpe by scraping. Spin down cells; remove all but10 mL of supernatant. Freeze (dry ice-ethanol bath) thaw cell pellet,vortex, re-freeze, and store virus stock in LN2 refrigerator.

ELISA Antibody Buffer: PBS, 0.05% Tween 20 (w/v), 2.0% goat serum (v/v)and 0.5% gelatin (w/v).

RSV F Protein Antibody: Chemicon Mab 858-1 anti-RSV fusion proteindiluted ˜1:5000 in ELISA Antibody Buffer.

Anti-Murine Serum.: Fisher horse radish peroxidase conjugated to goatanti-mouse IgG (Heavy Chain Specific) diluted ˜1:4000 in ELISA AntibodyBuffer.

The results are shown in FIG. 10, and indicate 25 ng/mi achieved 50%neutralization in this assay while 45 ug/ml of the ch1129 antibody wasrequired for 50% neutralization in this experiment. Over a series of 6separate assays the mean 50% neutralization value for H1129 was 17ng/ml. As a control and to compare potency we also assayed a polyclonalhuman IgG preparation made from the plasma of individuals with highneutralizing titers for RSV. This preparation, termed RSVig (lot#4),gave a mean 50% neutralization value of 2.3 ug/ml over 3 experiments.Thus the H1129 is 100-fold more potent in this assay as the enrichedpolyclonal preparation.

EXAMPLE 11

Kinetic Analysis of Humanized RSV Mabs by BlAcoreTM

The kinetics of interaction between humanized RSV Mabs and the RSV Fprotein was studied by surface plasmon resonance using a PharmaciaBlAcoreTM biosensor. A recombinant baculovirus expressing a C-terminaltruncated F protein provided an abundant source of antigen for kineticstudies. The supernatant, which contained the secreted F protein, wasenriched approximately 20-fold by successive chromatography onconcanalvalin A and Q-sepharose columns. The pooled fractions weredialyzed against 10 mM sodium citrate (pH 5.5), and concentrated toapproximately 0.1 mg/ml. An aliquot of the F-protein (100 ml) wasamine-coupled to the BlAcore sensor chip. The amount immobilized gaveapproximately 2000 response units (Rmax) Of signal when saturated witheither H1129 or H1308F. This indicated that there was an equal number of"A" and "C" antigenic sites on the F-protein preparation following thecoupling procedure. Two unrelated irrelevant Mabs (RVFV 4D4 and CMVH758) showed no interation with the immobolized F protein. A typicalkinetic study involved the injection of 35 ml of Mab at varyingconcentrations (25-300 nM) in PBS buffer containing 0.05% Tween-20(PBS/Tween). The flow rate was maintained at 5 ml/min, giving a 7 minbinding phase. Following the injection of Mab, the flow was exchangedwith PBS/Tween buffer for 30 min for determining the rate ofdissociation. The sensor chip was regenerated between cycles with a 2min pulse of 10 mM HCl. The regeneration step caused a minimal loss ofbinding capacity of the immobilized F-protein (4% loss per cycle). Thissmall decrease did not change the calculated values of the rateconstants for binding and dissociation.

The affinity of the various Mabs for binding to the F protein wascalculated from the ratio of the first order rate constant fordissociation to the second order rate constant for binding (K_(d)=k_(diss) /k_(assoc)). The value for k_(assoc) was calculated based onthe following rate equation:

    dR/dt=k.sub.assoc  Mab!R.sub.max -(k.sub.assoc  Mab!+k.sub.diss)R(1)

where R and Rmax are the response units at time t and infinity,respectively. A plot of dr/dt as a function of R gives a slope of(k_(assoc) Mab!+k_(diss))--Since these slopes are linearly related tothe Mab!, the value k_(assoc) can be derived from a replot of the slopesversus Mab!. The slope of the new line is equal to kassoc. Although thevalue of kdiss can be extrapolated from the Y-intercept, a more accuratevalue was determined by direct measurement of k_(diss). Following theinjection phase of the Mab, PBS/Tween buffer flows across the sensorchip. From this point, Mab!=0. Equation (1) thus reduces to:

    dr/dt=k.sub.dissr or dR/R=k.sub.diss dt                    (2)

Integration of equation (2) gives:

    ln(R.sub.0 /R.sub.t)=k.sub.diss t                          (3)

where R₀ /R_(t)) are the response units at time 0 (start of dissociationphase) and t, respectively. Lastly, plotting In(R₀ /R_(t)) as a functionof t gives a slope of kdiss.

    ______________________________________                                        Kinetic Constants for RSV Mabs                                                          ka(assoc)                                                                              kd(dissoc) t.sub.1/2 #                                                                        K.sub.d (k.sub.d /k.sub.a)                 Mab       M.sup.-1 sec.sup.-1                                                                    sec.sup.-1 (Hrs)                                                                              nM                                         ______________________________________                                        CH1129    5.0 × 10.sup.-5                                                                  7.5 × 10.sup.-5                                                                    2.6  1.5                                        H1129     4.9 × 10.sup.-4                                                                  6.9 × 10.sup.-5                                                                    2.8  1.4                                        M1129     3.5 × 10.sup.-4                                                                  4.0 × 10.sup.-4                                                                    0.48 11.4                                       M1308F    3.5 × 10.sup.-4                                                                  3.8 × 10.sup.-5                                                                    5.1  1.1                                        H1308F    2.2 × 10.sup.-4                                                                  5.5 × 10.sup.-5                                                                    3.5  2.5                                        ______________________________________                                    

References

1. Hall, C. B., Doiuglas, R. G., Geiman, J. M. et al., N.Engl.J.Med.293:1343, 1975.

2. Hall, C. B., McBride, J. T., Walsh, E. E. et al., N.Engl.J.Med.308:1443, 1983.

3. Hall, C. B., McBride, J. T., Gala, C. L. et al., JAMA 254:3047, 1985.

4. Wald, E. R., et al., J.Pediat. 112:154, 1988.

5. Kapikian, A. Z., Mithcell, R. H., Chanock, R. M. et al.,Am.J.Epidemiol. 89:405, 1969.

6. Prince, G. A., Hemming, V. G., Horswood, R. L. et al., Virus Res.3:193, 1985.

7. Hemming, V. G., Prince, G. A., Horswood, R. L. et al., J.Infect.Dis.152:1083, 1985.

8. Wright, P. F., Belshe, R. B., et al., Infect.Immun. 37:397, 1982.

9. Conrad, D. A., Christenson, J. C., et al., Peditr.Infect.Dis.J.6:152, 1987.

10. LoBuglio, A. F., Wheeler, R. L., Trang, J. et al., Proc.Natl.Acad.Sci. 86:4220, 1989.

11. Steplewski, Z., Sun, L. K., Shearman, C. W. et al., Proc.Natl.Acad.Sci. 85:4852, 1988.

12. Boulianne, G. L., Hozumi, N., Shulman, M. J. Nature. 312:643, 1984.

13. Sun, L. K., Curtis, P., Rakowicz-Szulczynska, E. et al.,Proc.Natl.Acad. Sci. 84:214, 1987.

14. Liu, A. Y., Mack, P. W., Champion, C. I., Robinson, R. R., Gene54:33, 1987.

15. Morrison, S. L., Johnson, M. J., Hersenber, L. A., Oi, V. T.Proc.Natl.Acad. Sci. 81:6851, 1984.

16. Morrison, S. L. Science 229:1202, 1985.

17. Sahagan, B. G., Dorai, H., Saltzgaber-Muller, J. et al., J.Immunol.137:1066, 1986.

18. Taked, S., Naito, T., Hama, K., Noma, T., Honjo, T., Nature 314:452,1985.

19. Carson, D. A., Freimark, B. D., Adv. Immunol. 38:275, 1986.

20. Beeler, J. A., et al., J.Virol. 63:2941-2950, 1989.

21. Coelingh, et al., Virology, 143:569-582, 1985.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 49                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGCGGATCCAGGGGCCAGTGGATAGAC27                                                 (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TGGATGGTGGGAAGATG17                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGCCAGTGGATAGAC15                                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TACAGTTGGTGCAGCA16                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GATGGATCCAGTTGGTGCAGCATC24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CACGTCGACATTCAGCTGACCCAGTCTCCA30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CGGAATTCAGGTNNANCTGCAGNAGTCWGG30                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCCAAGCTTGGTCCCCCCTCCGAACGTG28                                                (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGCGTCGACTCACCATGGACATGAGGGTCCYCGCTCAGC39                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GTCACCATCACTTGCAAGTGCCAGCTGAGTGTAGGTTACATGCACTGGTACCAGCAG57                   (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCAACTTATTACTGCTTTCAGGGGAGTGGGTACCCATTCACGTTCGGAGGGGGG54                      (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GTGACCAACATGGACCCTGCTGATACTGCCAC32                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCATGTTGGTCACTTTAAGGACCACCTGG29                                               (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCAGTTTACTAGTGTCATAGATCAGGAGCTTAGGGGC37                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TGACACTAGTAAACTGGCTTCTGGGGTCCCATCAAGG37                                       (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly                                 51015                                                                         AlaSerValLysValSerCysLysAlaSerGlyTyrThrPheAsn                                 202530                                                                        SerTyrTyrMetHisTrpValArgGlnAlaProGlyGlnGlyLeu                                 354045                                                                        GluTrpMetGlyIleIleAsnProSerGlyGlySerThrSerTyr                                 505560                                                                        AlaGlnLysPheGlnGlyArgValThrMetThrArgAspThrSer                                 657075                                                                        ThrSerThrValTyrMetGluLeuSerSerLeuArgSerGluAsp                                 808590                                                                        ThrAlaValTyrTyrCysAla                                                         95                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly                                 51015                                                                         AlaSerValLysValSerCysLysAlaSerGlyPheAsnIleLys                                 202530                                                                        AspTyrTyrIleTyrTrpValArgGlnAlaProGlyGlnGlyLeu                                 354045                                                                        GluTrpIleGlyTrpIleAspProGluAsnGlyAsnThrValPhe                                 505560                                                                        AspProLysPheGlnGlyArgValThrMetThrArgAspThrSer                                 657075                                                                        ThrSerThrValTyrMetGluLeuSerSerLeuArgSerGluAsp                                 808590                                                                        ThrAlaValTyrTyrCysAlaTyrTyrGlyThrSerSerPheAsp                                 95100105                                                                      PheTrpGlyGlnGlyThrThrLeuThrValSerSer                                          110115                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GluValGlnLeuGlnGlnSerGlyAlaGluLeuValArgProGly                                 51015                                                                         AlaLeuValLysLeuSerCysLysAlaSerGlyPheAsnIleLys                                 202530                                                                        AspTyrTyrIleTyrTrpValLysGlnArgProGluGlnGlyLeu                                 354045                                                                        GluTrpIleGlyTrpIleAspProGluAsnGlyAsnThrValPhe                                 505560                                                                        AspProLysPheGlnGlyLysAlaSerIleThrSerAspThrSer                                 657075                                                                        SerAsnThrAlaTyrLeuGlnLeuSerSerLeuThrSerGluAsp                                 808590                                                                        ThrAlaValTyrTyrCysAlaTyrTyrGlyThrSerSerPheAsp                                 95100105                                                                      PheTrpGlyGlnGlyThrThrLeuThrValSerSer                                          110115                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AspIleGlnMetThrGlnSerProSerThrLeuSerAlaSerVal                                 51015                                                                         GlyAspArgValThrIleThrCysArgAlaSerGlnSerIleSer                                 202530                                                                        SerTrpLeuAlaTrpTyrGlnGlnLysProGlyLysAlaProLys                                 354045                                                                        LeuLeuIleTyrAspAlaSerSerLeuGluSerGlyValProSer                                 505560                                                                        ArgPheSerGlySerGlySerGlyThrGluPheThrLeuThrIle                                 657075                                                                        SerSerLeuGlnProAspAspPheAlaThrTyrTyrCysGlnGln                                 808590                                                                        TyrAsnSerTyrSer                                                               95                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AspIleGlnMetThrGlnSerProSerThrLeuSerAlaSerVal                                 51015                                                                         GlyAspArgValThrIleThrCysLysAlaSerGlnAspIleAsn                                 202530                                                                        ArgTyrLeuAsnTrpTyrGlnGlnLysProGlyLysAlaProLys                                 354045                                                                        LeuLeuIleTyrArgAlaAsnArgLeuValAspGlyValProSer                                 505560                                                                        ArgPheSerGlySerGlySerGlyThrGluPheThrLeuThrIle                                 657075                                                                        SerSerLeuGlnProAspAspPheAlaThrTyrTyrCysLeuGln                                 808590                                                                        PheHisGluPheProTyrThrPheGlyGlyGlyThrLysLeuGlu                                 95100105                                                                      IleLys                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AspIleLysMetThrGlnSerProSerSerMetTyrValSerLeu                                 51015                                                                         GlyGluArgValThrIleThrCysLysAlaSerGlnAspIleAsn                                 202530                                                                        ArgTyrLeuAsnTrpPheGlnGlnLysProGlyLysSerProLys                                 354045                                                                        ThrLeuIleHisArgAlaAsnArgLeuValAspGlyValProSer                                 505560                                                                        ArgPheSerGlySerGlySerGlyGlnGluTyrSerLeuThrIle                                 657075                                                                        SerSerLeuGluPheGluAspMetGlyIleTyrTyrCysLeuGln                                 808590                                                                        PheHisGluPheProTyrThrPheGlyGlyGlyThrLysLeuGlu                                 95100105                                                                      IleLys                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CCATGGACTGGACCTGGAGGGTCTTCTGCTTGCTGGCTGTAGCACCAGGTGCCCACTCCC60                AGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCCTCAGTGAAGG117                  (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CACTTCTTCGGACCTCGGAGTCACTTCCAAAGGACGTTCCGTAGACCTAAGTTGTAATTC60                CTGATGATGTAAATGACCCACGCTGTCCGAGGACCTGTTCCCGAGCTCACCTACCCAACC120               (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GGGCTCGAGTGGATGGGTTGGATTGACCCTGAGAATGGTAATACTGTGTTTGACCGAAGT60                TCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTG119                (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 137 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGTGCTCGTGTCAGATGTACCTCGACTCGTCGGACTCTAGACTCCTGTGCCGGCACATAA60                TGACACGCATGATGCCATGTTCGAGGAAACTGAAGACCCCGGTTCCGTGGTGAGAGTGTC120               ACTCGAGTATTCCTAGG137                                                          (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 106 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTG60                CCAAATGTGATATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGC106                             (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GTCAGAGGAAGGTGGGACAGACGTAGACATCCTCTGTCTCAGTGGTAGTGAACGTTCCGC60                TCAGTCCTGTAATTATCCATAAATTTGACCATGGTCGTCTTTGGGCC107                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GAAAGCCCCTAAGCTCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAG60                GTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCA107                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 NUCLEOTIDES                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GTCTTAAGTGAGAGTGGTAGTCGTCGGACGTCGGACTACTAAAACGTTGAATAATGACGG60                ATGTCAAAGTACTCAAAGGCATGTGCAAGCCTCCCCCCTGGTTCGAACTTTATTTT116                   (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GlnValThrLeuArgGluSerGlyProAlaLeuValLysProThr                                 51015                                                                         GlnThrLeuThrLeuThrCysThrPheSerGlyPheSerLeuSer                                 202530                                                                        SerSerGlyMetCysValGlyTrpIleArgGlnProProGlyLys                                 354045                                                                        AlaLeuGluTrpLeuAlaAspIleGluTrpAspAspAspLysAsp                                 505560                                                                        TyrAsnThrSerLeuAspThrArgLeuThrIleSerLysAspThr                                 657075                                                                        SerLysAsnGlnValValLeuThrValThrAsnMetAspProAla                                 808590                                                                        AspThrAlaThrTyrTyrCysAlaArgIleThrValIleProAla                                 95100105                                                                      ProAlaGlyTyrMetAspValTrpGlyArgGlyThrProValThr                                 110115120                                                                     ValSerSer                                                                     (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GlnValThrLeuArgGluSerGlyProAlaLeuValLysProThr                                 51015                                                                         GlnThrLeuThrLeuThrCysThrPheSerGlyPheSerLeuSer                                 202530                                                                        ThrSerGlyMetSerValGlyTrpIleArgGlnProSerGlyLys                                 354045                                                                        AlaLeuGluTrpLeuAlaAspIleTrpTrpAspAspLysLysAsp                                 505560                                                                        TyrAsnProSerLeuLysSerArgLeuThrIleSerLysAspThr                                 657075                                                                        SerLysAsnGlnValValLeuLysValThrAsnMetAspProAla                                 808590                                                                        AspThrAlaThrTyrTyrCysAlaArgSerMetIleThrAsnTrp                                 95100105                                                                      TyrPheAspValTrpGlyAlaGlyThrThrValThrValSerSer                                 110115120                                                                     (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GlnValGluLeuGlnGluSerGlyProGlyIleLeuGlnProSer                                 51015                                                                         GlnThrLeuSerLeuThrCysSerPheSerGlyPheSerLeuSer                                 202530                                                                        ThrSerGlyMetSerValGlyTrpIleArgGlnProSerGlyGlu                                 354045                                                                        GlyLeuGluTrpLeuAlaAspIleTrpTrpAspAspLysLysAsp                                 505560                                                                        TyrAsnProSerLeuLysSerArgLeuThrIleSerLysAspThr                                 657075                                                                        SerSerAsnGlnValPheLeuLysIleThrGlyValAspThrAla                                 808590                                                                        AspThrAlaThrTyrTyrCysAlaArgSerMetIleThrAsnTrp                                 95100105                                                                      TyrPheAspValTrpGlyAlaGlyThrThrValThrValSerSer                                 110115120                                                                     (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AspIleGlnMetThrGlnSerProSerThrLeuSerAlaSerVal                                 51015                                                                         GlyAspArgValThrIleThrCysArgAlaSerGlnSerIleSer                                 202530                                                                        SerTrpLeuAlaTrpTyrGlnGlnLysProGlyLysAlaProLys                                 354045                                                                        LeuLeuIleTyrAspAlaSerSerLeuGluSerGlyValProSer                                 505560                                                                        ArgPheSerGlySerGlySerGlyThrGluPheThrLeuThrIle                                 657075                                                                        SerSerLeuGlnProAspAspPheAlaThrTyrTyrCysGlnGln                                 808590                                                                        TyrAsnSerTyrSer                                                               95                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 106 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      AspIleGlnMetThrGlnSerProSerThrLeuSerAlaSerVal                                 51015                                                                         GlyAspArgValThrIleThrCysLysCysGlnLeuSerValGly                                 202530                                                                        TyrMetHisTrpTyrGlnGlnLysProGlyLysAlaProLysLeu                                 354045                                                                        TrpIleTyrAspThrSerLysLeuAlaSerGlyValProSerArg                                 505560                                                                        PheSerGlySerGlySerGlyThrGluPheThrLeuThrIleSer                                 657075                                                                        SerLeuGlnProAspAspPheAlaThrTyrTyrCysPheGlnGly                                 808590                                                                        SerGlyTyrProPheThrPheGlyGlyGlyThrLysLeuGluIle                                 95100105                                                                      Lys                                                                           (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 106 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AspIleGlnLeuThrGlnSerProAlaIleMetSerAlaSerPro                                 51015                                                                         GlyGluLysValThrMetThrCysSerAlaSerSerSerValGly                                 202530                                                                        TyrMetHisTrpTyrGlnGlnLysSerSerThrSerProLysLeu                                 354045                                                                        TrpIleTyrAspThrSerLysLeuAlaSerGlyValProGlyArg                                 505560                                                                        PheSerGlySerGlySerGlyAsnSerTyrSerLeuThrIleSer                                 657075                                                                        SerIleGlnAlaGluAspValAlaThrTyrTyrCysPheGlnGly                                 808590                                                                        SerGlyTyrProPheThrPheGlyGlyGlyThrLysLeuGluIle                                 95100105                                                                      Lys                                                                           (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GCCTGAGCTCACGGTGACCGTGGTCCCGCCGCCCCAGACATCGAAGTAGCAGTTCGTGATCA63              (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GTTGGTGACTTTAAGGACCACCTGGTTTTTGGAGGTATCCTTGGAGATTGTGAGCCGGCT60                CTTCAGCCATGGATTATAG79                                                         (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 89 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      GCGCCTTCCCTGGGGGCTGACGAATCCAGCCTACACTCATACCAGAAGTGCTCAGTGAAA60                ACCCAGAGAAGGTGGAGGTCAGTGTGAGG89                                               (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CCAGGTCACCTTAAGGGAGTCTGGTCCTGCGCTGGTGAAACCCACACAGACCCTCACACT60                GACCTGCACC70                                                                  (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CAGCCCCCAGGGAAGGCCCTGGAGTCGCTTGCAGACATTTGGTGGGATGACAAAAAGGAC60                TATAATCCATCCCTGAAG78                                                          (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GGTCCTTAAAGTGACCAACATGGACCCTGCTGATACTGCCACTTACTACTGTGCTCGGTC60                TATG64                                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 NUCLEOTIDES                                                    (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GGCGTCGACTCACCATGGACTGGACCTGGAGGGTCTTCTGCTTGCTGGCTGTAGCACCAG60                GTGCCCACTCCC72                                                                (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 AMINO ACIDS                                                     (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ThrSerGlyMetSerValGly                                                         (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AspIleTrpTrpAspAspLysLysAspTyrAsnProSerLeuLysSer                              51015                                                                         (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      SerMetIleThrAsnTrpTyrPheAspVal                                                510                                                                           (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      LysCysGlnLeuSerValGlyTyrMetHis                                                510                                                                           (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 AMINO ACIDS                                                     (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      ThrSerLysLeuAlaSer                                                            5                                                                             (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 AMINO ACIDS                                                     (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      PheGlnGlySerGlyTyrProPhe                                                      5                                                                             (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 AMINO ACIDS                                                     (B) TYPE: AMINO ACID                                                          (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      SerValGlyTyrMetHis                                                            5                                                                             __________________________________________________________________________

What is claimed is:
 1. A neutralizing antibody against RSV, comprising:ahuman constant region and a variable region, said variable regioncomprising heavy and light chain framework regions and heavy and lightchain CDRs, at least a portion of the heavy and light chain frameworkregions being derived from a human antibody, said neutralizing antibodyagainst respiratory syncytial virus binding to the same epitope as anantibody comprising three heavy chain CDRs comprising amino acids 31-37,52-67 and 100-109 of SEQ ID NO:31, and three light-chain CDRs comprisingamino acids 24-33, 51-56 and 89-96 of SEQ ID NO:34.
 2. The neutralizingantibody of claim 1 wherein the heavy chain framework comprises theheavy chain framework of SEQ ID NO.
 31. 3. The neutralizing antibody ofclaim 1 wherein the light chain framework comprises the light chainframework of SEQ ID NO:34.
 4. The neutralizing antibody of claim 1wherein the light chain framework comprises the light chain framework ofSEQ. ID NO. 34 and the heavy chain framework comprises the heavy chainframework of SEQ ID NO:31.
 5. The neutralizing antibody of claim 1wherein the heavy chain of the neutralizing antibody comprises thepolypeptide of SEQ. ID NO:31.
 6. The neutralizing antibody of claim 1wherein the light chain of the neutralizing antibody comprises thepolypeptide of SEQ. ID NO:34.
 7. The neutralizing antibody of claim 1wherein the heavy chain of the neutralizing antibody comprises SEQ IDNO:31 and the light chain of the antibody comprises SEQ ID NO:34.
 8. Aneutralizing antibody against respiratory syncytial virus, comprising:ahuman constant region and a heavy and light chain variable region, saidheavy and light chain variable region comprising heavy and light chainframework regions and heavy and light chain CDRs, at least a portion ofthe heavy and light chain framework regions being derived from a humanantibody, said CDRs comprising three heavy-chain CDRs comprising aminoacids 31-37, 52-67 and 100-109 of SEQ ID NO:31 and three light-chainCDRs comprising amino acids 24-33, 51-56 and 89-96 of SEQ ID NO:34.
 9. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 1 and (b) a pharmaceutically acceptable diluent.
 10. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 2 and (b) a pharmaceutically acceptable diluent.
 11. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 3 and (b) a pharmaceutically acceptable diluent.
 12. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 4 and (b) a pharmaceutically acceptable diluent.
 13. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 5 and (b) a pharmaceutically acceptable diluent.
 14. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 6 and (b) a pharmaceutically acceptable diluent.
 15. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 7 and (b) a pharmaceutically acceptable diluent.
 16. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 8 and (b) a pharmaceutically acceptable diluent.
 17. A process fortreating or preventing respiratory syncytial virus infection in a human,comprising:administering to a human the neutralizing antibody ofclaim
 1. 18. A process for treating or preventing respiratory syncytialvirus infection in a human, comprising:administering to a human theneutralizing antibody of claim
 2. 19. A process for treating orpreventing respiratory syncytial virus infection in a human,comprising:administering to a human the neutralizing antibody of claim3.
 20. A process for treating or preventing respiratory syncytial virusinfection in a human, comprising:administering to a human theneutralizing antibody of claim
 4. 21. A process for treating orpreventing respiratory syncytial virus infection in a human,comprising:administering to a human the neutralizing antibody of claim5.
 22. A process for treating or preventing respiratory syncytial virusinfection in a human, comprising:administering to a human theneutralizing antibody of claim
 6. 23. A process for treating orpreventing respiratory syncytial virus infection in a human,comprising:administering to a human the neutralizing antibody of claim7.
 24. A process for treating or preventing respiratory syncytial virusinfection in a human, comprising:administering to a human theneutralizing antibody of claim
 8. 25. The neutralizing antibody of claim8 wherein the heavy chain framework comprises the heavy chain frameworkof SEQ ID NO.
 31. 26. The neutralizing antibody of claim 8 wherein thelight chain framework comprises the light chain framework of SEQ IDNO:34.
 27. A process for treating or preventing respiratory syncytialvirus infection in a human, comprising:administering to a human theantibody of claim
 25. 28. A process for treating or preventingrespiratory syncytial virus infection in a human,comprising:administering to a human the antibody of claim
 26. 29. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 25 and (b) a pharmaceutically acceptable diluent.
 30. Apharmaceutical composition comprising:(a) the neutralizing antibody ofclaim 26 and (b) a pharmaceutically acceptable diluent.